1. Field of the Invention (Technical Field)
The present invention relates to devices, systems and methods for electrochemical detection of specific binding pair interactions utilizing microspheres with incorporated electroactive markers, wherein one member of the specific binding pair is bound, directly or through one or more intermediates, to the surface of the microsphere.
2. Description of Related Art
Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-à-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Detection of analytes, and particularly detection of analytes wherein the analyte is a member of a specific binding pair, is well known in the art. A wide variety of bioassays are known which use a number of different reporting systems, such as fluorescent probes, radioactive markers and the like.
One specific binding pair that can be detected is DNA hybridization, used for diagnosis and treatment of genetic diseases, for the detection of infectious agents, for reliable forensic analysis and other purposes. DNA sensing applications require high sensitivity through amplified transduction of the oligonucleotide interaction. Electrical detection of DNA hybridization has shown great promise for this purpose and has thus been the subject of intense research activity. (Mikkelsen, S. R. Electroanalysis, 1996, 8, 15; Wang, J. Chem. Eur. J. 1999, 5, 1681; Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A; see also U.S. Pat. No. 6,387,625). Such electronic transduction is commonly accomplished by using intercalating electroactive indicators (that associate with the surface hybrid), through enzyme-amplified recognition, by monitoring the intrinsic redox activity of DNA, or through redox tags covalently bound to single-stranded DNA oligomers. In particular, the use of ferrocene-oligonucleotide conjugates has been shown extremely useful for monitoring DNA and RNA down to the femtomol level. However, the sensitivity of such systems has suffered because of lack of an adequate reporter or amplification system for detecting small quantities of analytes.
U.S. Pat. No. 6,087,748 discloses liposomes encapsulating an electroactive marker in a specific test device with a contact portion, an electrochemical measurement portion, and a liposome lysing portion with a liposome lysing agent. However, this approach is limited by the inherent fragility of phospholipid bilayer constructs and the limited size and carrying capacity of liposomes, as well as the permeability of liposome membranes.
There remains a need for a method of employing electrochemical detection of analytes, such as members of specific binding pairs, which provides a reporting and amplification system, and further optionally provides a method for detection of extremely low levels of analytes.